Therefore, we measured the intracellular ROS level in control and STM treated cells. of NAC, a ROS scavenger. This screening strategy allowed us to identify novel bioactive compounds focusing on ROS A-484954 rate of metabolism. Santamarine (STM) was identified as a novel ROS-based anticancer compound during the screening process. STM is definitely a sesquiterpene lactone compound present in numerous plant varieties such asInula heleniumInula japonica Magnolia grandiflora Ambrosia confertiflora, andSaussurea lappa in vitroin a dose-dependent manner as demonstrated in Number 1(a). The IC50 ideals were found to be around 45 and 43? 0.05). (b) NL-20, A549, and NCI-H1650 cells were treated with the indicated concentrations of STM in the presence or absence of 3?mM NAC for 24?h. Cells’ morphological changes were observed under a phase-contrast microscope and images were captured. (c) NL-20, A549, and NCI-H1650 cells were treated A-484954 with the indicated concentrations of STM for 24?h and live and dead cells were quantified using live/dead assay. Pretreatment of cells with NAC reversed STM-induced death in A549 cells. Data are indicated as mean SEM (= 3). Columns not posting the same superscript characters differ significantly ( 0.05). 3.2. STM Induces Apoptotic Cell Death in A549 Cells Live/deceased assay can be used to quantify live and deceased cells. To characterize the nature of cell death induced by STM, we performed the apoptosis assay by staining the cells with Annexin V-FITC/PI staining. The data shown that STM induces apoptotic cell death inside a dose-dependent manner (Number 2). STM-induced apoptosis was reversed by pretreatment of cells with 3?mM NAC mainly because shown in Number 2. Open in a separate window Number 2 STM induces apoptosis in A549 cells. (a) A549 cells were treated with 0 and 40? 0.05). 3.3. STM Induces ROS Generation in A549 Cells As demonstrated in Figures ?Numbers22 and ?and3,3, NAC (ROS scavenger as well while GSH precursor) effectively abrogated the STM-induced cell death in A549 cells. Consequently, we measured the intracellular ROS level in control and STM treated cells. No switch in intracellular ROS level was recognized after 24?h drug treatment. Therefore, we measured the level of ROS inside a time-dependent manner. The data showed that STM FAXF induces ROS generation inside a time-dependent manner. As demonstrated in Number 3, STM started to increase ROS generation as early as 1?h of treatment which reached its maximum level at 4?h and then started to decrease at 8?h. Next, we measured the level A-484954 of ROS inside a dose-dependent manner at 4?h drug exposure. The data showed that STM improved ROS generation inside a dose-dependent manner at 4?h drug treatment (Number 3(a)). Open in a separate window Number 3 STM induces oxidative stress in A549 cells. (a) A549 cells were treated with STM inside a dose- and time-dependent manner as indicated and intracellular ROS generation was measured by staining the cells with DCFH-DA. (b) A549 cells were treated with STM for 24?h in the presence or absence of NAC and intracellular GSH/GSSG percentage was measured according to the kit instructions. (c) A549 cells were treated with STM for 24?h in the presence or absence of NAC and TrxR activity was measured according to the kit instructions. Data (from (a), (b), and (c)) are indicated as mean SEM of three different experiments. Columns not posting the same superscript characters within the same group differ significantly ( 0.05). 3.4. STM Decreases GSH/GSSG Percentage in A549 Cells Intracellular GSH is the major antioxidant that helps prevent cells from ROS-mediated toxicity. Depletion of intracellular GSH level is an early hallmark in ROS-mediated apoptosis in multiple malignancy cells . GSH/GSSG percentage is critical for cellular redox potential. A decrease in GSH/GSSG percentage is a sensitive indication of oxidative stress. Therefore, we measured GSH/GSSG percentage. STM decreased intracellular GSH/GSSG percentage inside a dose-dependent manner (Number 3(b)). This GSH depletion was efficiently inhibited by NAC pretreatment. 3.5. STM Inhibits Intracellular Thioredoxin Reductase (TrxR) Activity Thioredoxin (Trx) is definitely another important antioxidant system of cells that protects the cells from your damaging effect of high ROS and maintains.